Enzymes have a role in every biological function and disease process. The ability to reliably design new enzyme catalysts would have major benefits for medicine and biomedical research. However, the structural features that make enzymes excellent catalysts are not yet fully understood. The proposed research seeks to understand how enzyme structure defines function. Specifically, a powerful combination of directed laboratory evolution and mechanistic analysis will be used to determine the sequence elements that define catalytic specificity in two related enzymes and to understand how they affect the catalytic function. Alkaline phosphatase catalyzes the hydrolysis of phosphate monoesters and has structural similarity to nucleotide pyrophosphatase/phosphodiesterase, an enzyme that catalyzes the hydrolysis of phosphate diesters. The two enzymes share only 16% sequence identity in a structurally conserved region, but they have almost identical zinc ion reaction centers. Because of the low sequence identity between the two proteins, it is impossible to determine which sequence differences lead to the differential catalytic activities. Directed evolution will be used to increase the existing weak promiscuous catalytic activity of nucleotide pyrophosphatase/phosphodiesterase toward phosphate monoesters. This evolutionary process will make it possible to identify sequence changes associated with increased monoesterase acivity. These sequence changes will be investigated in depth by studying the effect of substrates with varied effective charge at the breaking bond. These analyses will make it possible to investigate whether or not the sequence changes affect the transition state structure for the reaction, how the dinuclear zinc reaction center discriminates between substrates based on their charge, and how the electrostatic environment near the zinc sites affects their catalytic specificity. It is expected that the project will show how specific sequence changes can lead to dramatic changes in catalytic activity. Relevance: Enzymes have a role in every biological function and disease process, but their mechanisms of action are not fully understood. Studying the mechanistic effects of changes in enzyme structure permits greater understanding of these essential biological catalysts that may permit the development of new enzymes with therapeutic and diagnostic value.